Many pathways have been implicated in heart disease, but less is understood about what keeps the heart healthy. Clinical and experimental studies document the impact of exercise in both preventing and treating heart disease, including ischemic injury and heart failure (HF). Prior work from our laboratory identified a distinct physiological gene set induced by exercise, as well as key transcription factor nodes in this network. Remarkably, exercise also activated a proliferative and potentially regenerative gene program in the heart. Genetically mimicking exercise-induced changes was sufficient to recapitulate many phenotypic features of the exercised heart, as well as to protect against HF. Like transcription factors, micro-RNAs (miRs) also regulate large gene expression networks and modify complex cardiovascular phenotypes but little is known about their role in exercise. We recently performed a comprehensive screen for miRs differentially expressed in hearts of exercised mice. Sixteen miRs were identified and validated as differentially regulated in exercised hearts, and then subjected to in vitro and in vivo functional assays. Our preliminary data demonstrate that cardiac miR-222 is robustly upregulated in two independent exercise models and appears to drive a proliferative response in cardiomyocytes (CMs). The overall goals of this R21 are to examine the functional roles of miR-222 in CMs in vitro and in vivo, and determine the downstream mechanisms. We hypothesize that exercise induces intrinsic signaling changes that promote cardiac health, in part by promoting regeneration, and that miR-222 is an important contributor to these benefits. We will use a combination of genetic and pharmacological interventions to test these hypotheses in vivo, while identifying the responsible mechanisms. If successful, we anticipate that these studies will provide new insights into the benefits of exercise and pathways controlling the regenerative capacity of the heart. In addition, the unique animal models generated will provide a foundation for validating miR-222 as a therapeutic target in cardiac disease.